Process for preparing 1-{62 -d-arabinofuranosyl-cytosine and 2,2{40 -cyclocytidine

ABSTRACT

The present invention relates to a novel process for preparing 1- Beta -D-arabinofuranosylcytosine (spongocytidine) expressed by the general formula (I) and a process for preparing as an intermediate thereof 2,2&#39;&#39;-cyclocytidine(2,2&#39;&#39;-anhydro-1- Beta -Darabinofuranosylcytosine) (II) with good efficiency.

United States Patent 1 Kanai et a1.

[ Aug. 28, 1973 PROCESS FOR PREPARING 1-B-D-ARABINOFURANOSYL-CYTOSINEAND 2,2'-CYCLOCYTIDINE [73] Assignee: Kohjin Co., Ltd., Tokyo, Japan[22] Filed: July 17, 1970 [2]] Appl. No.: 55,966

[30] Foreign Application Priority Data July 24, 1969 Japan 44/58517 Dec.4, 1969 Japan 44/97482 Dec. 4, 1969 Japan 44/97483 Mar. 24, 1970 Japan45/24744 [52] US. Cl 260/2115 R [51] Int. Cl C07d 51/52. [58] Field ofSearch 260/2115 R [56] References Cited UNITED STATES PATENTS 3,155,64611/1964 Hunter 260/2115 R 3,463,772 8/1969 Nagyvary 260/2115 R 3,595,8537/1971 Kanai et a1 260/2115 R FOREIGN PATENTS OR APPLICATIONS 4,313,2146/1968 Japan 260/2115 OTHER PUBLICATIONS Wolfrom, Advances inCarbohydrate Chem., Vol. 22, 1967, Academic Press, New York, N.Y., pp.350- Wolfrom, Advances in Carbohydrate Chem", V01. 24, 1969, AcademicPress, New York, N.Y., pp. 180-184.

Primary Examiner--Lewis Gotts Assistant Examiner-Johnnie R. BrownAttorney-McGlew and Toren [57] ABSTRACT The present invention relates toa novel process for preparing 1-B-D-arabinofuranosylcytosine(spongocytidine) expressed by the general formula (1) and a process forpreparing as an intermediate thereof 2,2-cyc1ocytidine(2,2'-anhydro-1-B-D- arabinofuranosylcytosine) (11) withgood efficiency.

NH, H

H z O I Home 0 (L K a K 1 J in 17 Claims, No Drawings 1 PROCESS FORPREPARING l-B-D-ARABINOFURANOSYL-CYTOSINE AND 2,2'-CYCLOCYTlDlNE Theknown processes for preparing l-B-D- arabinofuranosylcytosine includethe following four processes.

1. The process of synthesis via a l-B-D- arabinofuranosylthiouracilderivative which is produced in four stages starting from l-B-D-arabinofuranosyluracil that is obtained in five stages from uracil.(Evans and others; Proceedings of the Society of Experimental Biologyand Medicine, 106, 350 (1961) and US. Pat. No. 3,116,282).

2. The process in which N,O -triacylcytidine is prepared from cytidinein 3 stage reactions. Then the 2-position is tosylated and the productis kept standing in water at room temperature and finally it isdeacylated. (Reese and others; Tetrahedron Letters, 3499 (1966)).

3. The process in which 2,2'-cyclocytidine-3',5- diphosphate, obtainedas by-product of the reaction of cytidine with polyphosphoric acid, ishydrolyzed by alkali and further by phosphatase. (Walwick and others;Proceedings of the Chemical Society, 84 (1959); Journal of OrganicChemistry, 32, 816 (1967)).

4. The process in which 2,3,5-tri-O- benzylarabinofuranosylchloride iscondensed with the 2,4-dimethoxypyrimidine, sodium salt of4-methylthiopyrimidine-Z-one, and mercury salt of N- acetylcytosine,producing, respectively, 1-(2',3',5'-tri-O-benzyl-B-D-arabinofuranosyl)-4-methoxy-2( 11-1)- pyrimidinone, 1-( 2',3 ,5 '-tri-O-benzyl-B-D- arabinofurasyl)-4-methylthiopyrimidine-2-one,and, N-acetyll -(2',3 ,5 '-tri-O-benzyl-B-D- arabinofuranosyl)-cytosineand the former two are made to react with ammonia and the latter one ishydrolyzed with alkali followed by reduction. (Shen and others; Journalof Organic Chemistry, 30, 835 (1965)).

The processes in (l), (2) and (4) contain faults in that they requiremany stages of reactions and, if starting from a readily availablematerial, extremely complicated procedures have to be followed and theyields are remarkably low. More particularly, in the process of (2) theintermediate material,K 15 qfiacylcytidine, is difficult to synthesizeand in the process of (4) the condensation reactions of 2,3,5-tri-O-benzylarabinofuranosyl chloride with bases are of low yield. In theprocess of (3), however, the object material can be synthesized withrelative ease from cytidine, but an enzyme (phosphatase) is required inthe process.

The process inventors have already developed a process for preparing thementioned substance employing nitric esters of cytidine as anintermediate (U.S. Pat., Ser. No. 812,367 now US. Pat. No. 3,595,853),but the present invention provides a profitable method that far exceedsthe foregoing ones.

In considering that the previous process for preparing1B-D-arabinofuranosylcytosine was very complicated and of a low yield,the present inventors have investigated and succeeded in the firstprocess of this invention, in which the object material could beisolated with a high yield of about 70 percent without isolating norpurifying the intermediate 2,2'-cyclocytidine that was obtained fromcytidine in a single stage of process.

The first process of this invention relates to a process for directlypreparing 1-B-D-arabinofuranosylcytosine in a single stage of operationwithout isolating the inter mediary 2,2-cyclocytidine by reactingcytidine with a halomethylenedimethylammonium halide having the generalformula,

where X stands for C1 or Br.

1n carrying out the first process of this invention, cytidine isdissolved in a solution of halomethylenedimethylammonium halide in asolvent such as dimethylformamide, and the mixture is then stirred atroom temperature for several hours to form an intermediate2,2'-cyclocytidine. By adding this mixture to water and adjusting the pHto 9 to 12 with an alkali, such as, sodium hydroxide, potassiumhydroxide, concentrated ammonia, sodium alcoholate and potassiumalcoholate, the object material can be produced by hydrolysis. Theprocess of this invention is thus characterized by preparing1-B-D-arabinofuranosylcytosine with a good yield in a single stage ofoperation starting from cytidine, because the process of this inventiondoes not involve isolation and purification of the intermediate,2,2'-cyclocytidine, but rather involves hydrolyzing the reactionsolution. The halomethylenedimethylammonium halide used in the processof this invention can be synthesized by reacting dimethylformamide atroom temperature for from several to ten minutes in an organic solventwith an equimolar amount of a halogenating agent such as thionylchloride, thionyl bromide, phosphorus oxychloride, phosphorusoxybromide, phosphorus trichloride, phosphorus tribromide, phosgene,phosphorus pentachloride, phosphorus pentabromide and chlorine. Ahalomethylenedimethylammonium halide synthesized in any of the knownmethods may be used in the present invention. This reagent obtained fromthe reaction of dimethylformamide with a halogenating agent, eitherisolated or not may also be used. The reaction medium to be used in thepresent invention is selected from a group of solvents which dissolvecytidine and other reagents without decomposition, and dimethylformamidewhich is a component material for preparing the reagent is mostconveniently used as the reaction solvent. 1n the process of thisinvention, the amounts of reagents should be present in a molar ratio of2 to 20 times that of cytidine, the reaction temperature 10 to 30C andthe time of reaction from 1 to 10 hours.

The 1-B-D-arabinofuranosylcytosine produced by the process of thisinvention is prepared as follows: the reaction mixture is added towater, the pH is adjusted to 9 to 12, kept at this value for severalminutes and then adjusted to pH 2.0, the nucleoside is absorbed onto 5to 30 times as much active charcoal, which is washed with a hydrochloricacid solution to remove salt, washed with water, and then after the pHof the washings rises above pH 4.0, the nucleoside on the charcoal iseluted with a mixed solution of 20 to percent aqueous methyl alcohol andpyridine or concentrated ammonia in a volume ratio of 4 1. The eluentcontains l-B-D-arabinofuranosylcytosine and a small amount of unreactedcytidine.

To remove the remaining cytidine, a column of an anion exchanging resin,for example Dowex-l X 2 (borate type), is used to adsorb cytidine alone.Alternatively, column chromatography with an Ol-l-type anion alcoholmixed solution, and fractionation with Dowexl X 2 to obtain1-fi-D-arabino-furanosylcytosine.

The first and the second processes of this invention can be expressed bythe following equations:

exchanging resin such as, for example, Dowex l X 2 (OH-type) is used andthe remaining cytidine is eluted with 30 percent aqueous methyl alcoholand then then 1-B-D-arabinofuranosylcytosine is eluted with a 0.1Msolution of ammonium bicarbonate to obtain a pure product of the latter.(Dekker and others, Journal of the American Chemical Society, 87, 4027(1965)).

The second process of this invention is characterized by synthesizing1-B-D-arabinofuranosylcytosine by the process, in which cytidine istreated with partially hydrated phosphorus oxychloride, or partiallyhydrated phosphorus oxybromide, or a mixture of a tertiary alcohol suchas tert-butyl alcohol with phosphorus oxychloride, or a mixture oftertiary alcohol such as ten-butyl alcohol with phosphorus oxybromide,or dichlorophosphoric acid to derive 2,2'-cyclocytidine, and this isthen hydrolyzed with an alkali such as sodium hydroxide, potassiumhydroxide, concentrated ammonia, sodium alcoholate and potassiumalcoholate to obtain the final product. The solvent in which the abovereactions are performed, may be freely selected, so long as the solventdoes not unfavorably influence the reactions, and esters of acetic acidare most favourably used.

The most predominant feature of this invention consists in producing1-,8-D-arabinofuranosylcytosine in one step from cytidine, because theprocess of this invention, without obtaining any acyl compound, produces2,2 '-cyclocytidine as intermediate which, instead of being isolated, ishydrolyzed directly in the solution to obtainl-B-D-arabinofuranosylcytosine with a good yield. More particularly, theprocess proceeds as follows: cytidine is suspended in an ester of aceticacid such as, for example, ethyl acetate, which is then refluxed for 1to 2 hours with excessive dichlorophosphoric acid, or for 40 minutes to2 hours either with a mixed solution of 5 to times (molar ratio) as muchphosphorus oxychloride and the same volume of t-butyl alcohol or with amixed solution of 5 to 20 times (molar ratio) as much phosphorusoxybromide with the same volume of t-butyl alcohol, or refluxed for 40minutes to 2 hours either with 5 to 20 times (molar ratio) as muchpartially hydrated phosphorus oxychloride or with 5 to 20 times (molarratio) as much partially hydrated phosphorus oxybromide, to obtain2,2cyclocytidine.

The solution is then distilled to remove the solvent without isolatingthe intermediate substance, and the remaining residue is treated with analkali, such as, sodium hydroxide, potassium hydroxide, concentratedammonia, sodium alcoholate, and potassium alcoholate to hydrolyze theanhydronucleoside at pH 9 to 12. This is treated, as described before,successively by adsorption on active charcoal, elution with an ammonial8-D-arabinofuranosylcytosine prepared in the first and the secondprocesses of this invention can be made in the form of any salt type byadding an equivalent or slight excess of moles of an acid to its watersolution, evaporating to dryness and recrystallizing from ethanol. Thus,the following salts of this compound can be prepared salts of inorganicacids such as hydrochloric, sulfuric and nitric acids, and salts oforganic acids such as formic, acetic, sulfosalicylic and erotic acids.

2,2'-cyclocytidine, obtained as an intermediate in the process of thisinvention, is an important material in synthesizing medicines such asarabinofuranosylcytosine and other substances related to nucleic acid.

So far, 2,2'-cyclocytidine has not been prepared in a simple processbecause of its instability and the consequent difficulties in isolation.2,2'-cyclocytidine has been prepared by the following three processes:

1. ER. Walwick and others (Proceedings of the Chemical Society, 84(1960)) reacted cytidine with polyphosphoric acid and the by-product2,2'-cyclocytidine-3', 5'-diphosphate was treated with an enzyme,phosphatase, to obtain the object substance. ln this process, however,the disadvantages are the need of an enzyme, complicated operations offractionation and purification and the low yield.

2. LL. Doerr and others (Journal of Organic Chemistry, 32, 1462 (1967))used as starting material 2'- chlorocytidine which was produced in 10stages of operation from uridine, to obtain the object substance.

3. W.V. Ruyle and others (Journal of Medicinal Chemistry, 10, 331 (1967)on the other hand, used 2,4-dithio-2',3',5'-triacetylarabinofuranosyluracil as a starting material which wasproduced in 7 stages of operation to obtain the object substance.

The processes (2) and (3), however, could not be operated easily, sincethey require multistage operation for the synthesis.

As a result of extensive investigations to improve the previousprocesses for preparing 2,2'-cyclocytidine with regard to theircomplexity and low yield, the present inventors have succeeded inpreparing and isolating 2,2'-cyclocytidine in a single stage ofoperation from cytidine with ease and a high yield (approximatelypercent), and hence thus enhancing the value of the compound.

The first process of this invention for preparing 2,2 cyclocytidinecomprises reacting cytidine with a halomethylenedimethylammonium halideexpressed by the general formula,

where X stands for C1 or Br.

In carrying out the process of this invention, cytidine is dissolved ina solvent such as dimethylformamide containing ahalomethylenedimethylammonium halide and the mixture is stirred forseveral hours at a temperature near room temperature to obtain theobject substance.

The halomethylenedimethylammonium halide to be used in the invention canbe prepared by reacting dimethylformamide in an organic solvent for fromsevera] to ten minutes at approximately room temperature with anequimolar amount of a halogenating agent, such as, thionyl chloride,thionyl bromide, phosphorus oxychloride, phosphorus oxybromide,phosphorus trichloride, phosphorus tribromide, phosgene, phosphoruspentachloride, phosphorus pentabromide and chlorine. Thehalomethylenedimethylammonium halide for use in this invention may besynthesized in any of the known methods, and further this reagent may ormay not be isolated from the reacting mixture of dimethylformamide andhalogenating agent. Any solvents which dissolve cytidine and the reagentwithout decomposition can be used for the solvent in which the reactionof this invention proceeds, but dimethylformamide which is a componentmaterial for preparing the reagent is most conveniently used as thereaction solvent.

The 2,2|'-cyclocytidine which is formed in the process of this inventioncan be isolated from the reaction solution in the following manner: thesolution is poured into water to decompose the reagent and the productis adsorbed on Dowex-50(pyridinium type) and eluted with a formatebuffer solution containing pyridine. If the fraction containing theobject substance is evaporated to dryness, 2,2'-cyclocytidine isobtained as formate, while, if a water solution of the dried residue ismade to flow through Dowex-1(Cl type) and the eflluent solution isevaporated to dryness. 2,2-cyclocytidine is obtained as hydrochloricacid salt.

The second process of this invention for preparing 2,2'-cyclocytidinewith ease and high yield comprises adding cytidine for dissolution topartially hydrated phosphorus oxychloride, or partially hydratedphosphorus oxybromide, or a mixed solution of phosphorus oxychloride orphosphorus oxybromide in a tertiary alcohol such as tert-butyl alcohol,or dichlorophosphoric acid, and carrying out the reaction by heating for40 minutes to 2 hours.

For the above described solvent in which the reaction proceeds, aceticacid ester such as ethyl acetate and others may be employed so long asthey do not affect unfavorably the reaction.

As for the amounts of halogenating agents to be used in the process ofthis invention, partially hydrated phosphorus oxychloride and partiallyhydrated phosphorus oxybromide are used in a molar ratio of 5 to 20times as much as the cytidine. Similarly mixed solutions of tertiaryalcohol with phosphorus oxychloride or with phosphorus oxybromide (theratio of mixing being 1 l in volume) are prepared in the same molarratio, that is from 5 to 20 times as much as cytidine.Dichlorophosphoric acid is added in an amount from 5 to times (molarratio).

2,2'-cyclocytidine prepared in the process of this invention can beisolated as follows: water is added directly to the reaction mixture todecompose the excessive reagent, and the resulting solution is treatedwith Dowex-50 (I-l type) and the adsorbed substances, after beingthoroughly washed with water, is eluted with a sodium chloride solutionor a formate buffer solution containing pyridine. 2,2'-cyclocytidine isobtained by drying directly or, if needed, by drying after desalting thefraction which contains the compound.

2,2'-cyclocytidine prepared in the first and the second processes ofthis invention can be made in the form of any salt type by changing thetype of the ion exchanging resin. Thus, the following salts of thiscompound can be prepared: salts of inorganic acids such as hydrochloric,sulfuric and nitric acids, and salts of organic acids such as formic,acetic sulfosalicylic and orotic acids.

These facts will be explained in detail by referring to examples below.

EXAMPLE 1 Thionyl chloride (3ml) was added to dimethylformamide (20ml)and the mixture was left standing for 10 min. at 30C. Cytidine (2.0g)was added to it and allowed to react for 5 hours under stirring at roomtemperature. The reaction solution was poured into water (about 200ml)and sulfurous acid was removed by agitation and then the pH was adjustedto 9 with ammonia. After being kept for 10 min. at room temperature, thesolution was brought again to pH 2 with hydrochloric acid and treatedwith active charcoal (20g). This was thoroughly washed with water,eluted with 50 percent ethanol containing 2 percent ammonia. The eluentwas concentrated to dryness and treated with a 50ml column of Dowex l X2 (OH type). After the unreacted cytidine was removed with 30 percentmethyl alcohol, l-B-D-arabinofuranosyl cytosine was eluted with a 0.1Msolution of ammonium bicarbonate. The eluent was concentrated todryness, and crystals of the l-B-D- arabinofuranosyl-cytosine wereobtained from ethanol.

.Yield 1.40g (70.0 percent) m.p. 213 214 (decomp.) (a) +l58 (C 0.5 in HO), UV;A,, (pl-l 1) 282m;:., A ,(pH 7) 272m;;., Amin(pH 1) 242mg,Amin(pl-l 7) 25lmu.

Paper chromatography (Solvent: a mixture consisting of 5M ammoniumacetate (pH 9.5), saturated solution of sodium borate, ethyl alcohol and0.5M EDTA in the EXAMPLE 2 Thionyl bromide (3m!) was added todimethylformamide 20ml) and the mixture was allowed to stand for 30min.at 30C. Cytidine (2.0g) was added to it and was allowed to react for 5hours under stirring at room temperature. The same treatment as inExample 1 was followed to obtain l-B-D-arabinofuranosylcytosine. Yield:1.20g (60 percent). Physical properties of the crystals were the same asthose in Example 1.

EXAMPLE 3 To phosphorus pentachloride (2g) dissolved in carbontetrachloride (20ml) was added dimethylformamide (0.8g). Precipitateproduced was filtered off and washed with a small amount of carbontetrachloride and the residue was dissolved in dimethylformamide (Sml).Cytidine (1.0g) was added to this solution and' EXAMPLE 4 Water (7.6ml)was added to ice-cooled phosphorus oxychloride (38.7ml) and stirred for30min. This solution was added to ethyl acetate (500ml) in whichcytidine (5g) had been suspended. The whole mixture was refluxed for 2hours. The reaction solution was cooled and evaporated under reducedpressure. To the residue, a 2N solution of sodium hydroxide (1.15 liter)was added and kept at the room temperature for 24 hours under stirring.Subsequently, the solution was adjusted to pH 2 with concentratedhydrochloric acid (50ml), and condensed down to 250ml under reducedpressure. This solution was introduced into a column of active charcoal(60g) to adsorb nucleoside and the column was washed with a dilutehydrochloric acid solution of pH 2 until inorganic salts were no longerfound in the eluate. Then elution was continued with water to make pH ofthe eluate larger than 4. Finally a mixed solution (1 liter) of methylalcohol and ammonia in the ratio 4 l was used as eluent to elute thenucleosides. Theeluate was condensed to 100ml under reduced pressure,and then treated with a column of 60ml Dowex 1 X 2 (borate type). Theportion of the solution containing the solutes which were not adsorbedand the eluate (400ml) obtained when eluted with water were gathered.Crystals were separated when the solution was evaporated to drynessunder reduced pressure.

Methyl alcohol (ml) was added to the lumps of crystals to crash them.After being filtered, the crystals were washed with methyl alcohol anddried to obtain 1-fi-D-arabinofuranosylcytosine. Yield: 1.85g (37percent). Physical properties were the same as those in Example 1.

EXAMPLE 5 A mixed solution of phosphorus oxychloride (38.7rnl) andt-butyl alcohol (38.7ml) was added to ethyl acetate (500ml) in whichcytidine (5g) had been suspended and the whole mixture was stirred for1.5 hours at 60C, and then refluxed for 40 min.

After being cooled, the reaction solution was mixed with a 2N sodiumhydroxide solution (1.15 liters) and allowed to stand for 24 hours atthe room temperature. Subsequently, pH was adjusted to 2 withconcentrated hydrochloric acid (50ml) and the solution was condensed to250ml under reduced pressure. Following the same procedure as in Example4, l-fl-D- arabinofuranosylcytosine was obtained. Yield: 2.71 g (54percent). Physical properties coincided with those of Example 1.

EXAMPLE 6 Dichlorophosphoric acid (10g) was added to ethylacetate(100ml) in which cytidine (1.0g) had been suspended and themixture was refluxed for 1 hour. After being cooled, the reactionsolution was concentrated and the residue produced was dissolved in a 2Nsolution of potassium hydroxide, adjusted to pH 11.5, kept standing for24 hours at room temperature, brought to pH 2.0 with concentratedhydrogen chloride solution and finally concentrated down to 50ml underreduced pressure.

The resulting solution was treated with a column of active charcoal(12g) to adsorb the nucleosides, and the column was washed first withdilute hydrochloric acid of pH 2 until inorganic salts were no longerfound in the eluate and then with water until the pH of the eluatereached 4 or more. Then a mixed solution (200ml) of methyl alcohol andammonia in the ratio of 4 1 was introduced into the column in order todissolve out the nucleosides. The eluate was concentrated to a volume of20ml, and introduced into a column of 24m] Dowex l X 2 (OH type) toadsorb the nucleosides. A 30 percent solution of methyl alcohol (5liters) was introduced to elute cytidine out selectively and a 0.1Msolution of ammonium bicarbonate (2 liters) was introduced. The eluatewas concentrated and the residue was mixed with alcohol, and evaporatedagain to dryness at the bath temperature 40C under reduced pressure. Theresidue formed was dissolved in a small amount of methyl alcohol, leftstanding for 24 hours, then crystals of 1-B-D-arabinofuranosylcytosinewere separated. Yield: 0.27g (27.0 percent). Physical properties werecoincident with those of Example 1.

EXAMPLE 7 Thionyl chloride (3ml) was added to dimethylformamide (20ml)and kept standing for 10 min. at 30C. The solution was mixed withcytidine (2.0g) and allowed to react for 5 hours under stirring at theroom temperature. The reaction solution was poured into 200ml of water,stirred to expel sulfurous acid and introduced into a column of mlDowex-SOW X 4 (pyridinium type). The adsorbed species was eluted with a0.1M formic acid-pyridine buffer solution (pH 5.0). The fractioncontaining the product was dried under reduced pressure, and crystals of2,2- cyclocytidine (formate) were obtained from methanol. Yield: 1.1g(45 percent). m.p. 173 178 (decomp.). U.V. (pH 1 5); max 231, 263mu,ltmin 218, 243mg" EXAMPLE 8 Thionyl bromide (3ml) was added todimethylformamide (20ml) and was kept standing for 10min. at 30C.Cytidine (2.0g) was added to it and the mixture was treated in the samemanner as in Example 7 to obtain the crystals of 2,2'-cyclocytidine(formate). Yield: 1.0g (40 percent). Physical properties were coincidentwith those of Example 7.

EXAMPLE 9 To carbon tetrachloride (20ml) containing dissolved phosphoruspentabromide (2g) was added dimethylformamide (0.8g). Precipitateproduced was separated by filtration and washed with carbontetrachloride. The residue was dissolved in dimethylformamide (Sml) andcytidine (lg) was added to it. Following the same procedure as that ofExample 7, this solution was treated to obtain 2,2'-cyclocytidine(fonnate). Yield: 0.50g (40 percent). Physical properties werecoincident with those in Example 7.

EXAMPLE 1O Thionyl chloride (3ml) was added to dimethylformamide (20ml)and was kept standing for 10 min. at 30C. Cytidine (2.0g) was added toit and allowed to react for 5 hours under stirring at the roomtemperature. The reaction solution was poured into water (200ml) andstirred to expel sulfurous acid, and then transferred to a column of100ml Dowex-SOW X 4 (pyridinium type). The adsorbed species were elutedwith a 0.1M formic acid-pyridine buffer solution (pH 5.0).The fractioncontaining the product was dried under reduced pressure, dissolved againin water (50ml), and introduced into a column of Dowex- 1 X 2 (Cl type)(25ml). The eluate was condensed to dryness and crystals of the2,2'-cyclocytidine (hydrochloride) were obtained from ethanol. Yield:1.5g (65%). m.p. 258 26l (decmp.). (a) :22.02 (C: 2.0 in H O). UV (pH 15): A max 231 (e: 9700), 263.5 (10700) my; )tmin 217.5 (7,000), 243.5(6600) my" The melting point, the optical rotation and the ultravioletspectrum of this compound were coincident with those of the standardsample.

EXAMPLE 1 1 To ice-cooled phosphorus oxychloride (75.5ml) water (14.8m1)was added and the mixture was stirred for 2 hours. This solution wasthen added to ethyl acetate (1.0 liter) in which cytidine (g) had beensuspended and the resulting mixture was heated for min. at 50 to 70C,followed by refluxing for 2 hours. The reaction solution, after beingcooled, was concentrated and the residue formed was dissolved in 500mlof water, introduced into a column of 700ml Dowex- 50W (l-l type) whichwas then washed thoroughly with 16 liters of water. The adsorbed matterswere eluted with 30 liters of a 2 percent solution of sodium chlo ride.The fraction containing the object substance was concentrated down to500ml under reduced pressure. Sodium chloride separated was removed byfiltration and the filtrate was concentrated to dryness. The residue wasextracted with 50 percent ethanol and the ethanol was then distilledout. The remaining solution was treated again with a column of 250mlDowex-50W (H type). The adsorbed matter was eluted with a 0.5N formicacid-pyridine buffer solution and evaporated to dryness. A watersolution of the residue was passed through a column of 200ml Dowex-l (Cltype) and then concentrated. Crystallization from water gave 7.85g(yield: 77.88 percent) of 2,2'-cyclocytidine (hydrochloride). Physicalproperties were coincident with those of Example 10.

EXAMPLE 12 A mixture of phosphorus oxychloride (38.7ml) and t-butylalcohol (38.7ml) was added to ethyl acetate (500ml) in which cytidine(5g) had been suspended, and the resulting mixture was stirred for 1.5hours at 60C followed by refluxing for 40 min. After being cooled, thereaction solution was concentrated under reduced pressure and theresidue was dissolved in 250ml of water. The solution was treated with acolumn of 350ml Dowex-50W (l-l type) which was washed with 8 liters ofwater. Elution was conducted with a 0.5N formic acid-pyridine buffersolution and the fraction containing the object substance wasconcentrated to dryness to obtain 2,2'-cyclocytidine (formate). Physicalproperties were coincident with those obtained in Example 7.

EXAMPLE 13 To ethyl acetate (100ml) in which cytidine (1 g) had beensuspended dichlorophosphoric acid (10g) was added and refluxed for 1hour. The reaction solution after being cooled was concentrated todryness. The residue formed was dissolved in 50ml of water andintroduced into a column of ml Dowex-50W (H type) which was then washedwith water and treated as in Example 12 to give 2,2'-cyclocytidine(formate). Yield: 0.82g (73.34 percent). The melting point and otherphysical properties were coincident with those obtained in Example 7.

EXAMPLE 14 Water (1.52ml) was added to ice-cooled phosphorus oxybromide(22.96g) and stirred for 30 min. This solution was added to ethylacetate (ml) in which cytidine (1.0g) was suspended and refluxed for 2hours. The reaction solution, after being cooled, was concentrated todryness. The residue formed was dissolved in water and introduced into acolumn of 70ml Dowex- 5OW (H type) which was washed with water and thentreated as in Example 1 l to give 0.462g of 2,2- cyclocytidine(hydrochloride). Yield: 42.78 percent. Physical properties werecoincident with those obtained in Example 10.

I claim:

1 A process for preparing 1 B-D- arabinofuranosylcytosine or aninorganic acid salt or organic acid salt thereof consisting essentiallyof reacting cytidine, a mineral acid salt thereof, or a cytidine salt ofan acid selected from the group consisting of formic acid, acetic acid,sulfosalicylic acid and crotic acid with at least one material selectedfrom the group consisting of (a) a halomethylenedimethylammonium halideexpressed by the general formula where X, and X, stand for the same ordifferent halogen atoms and (b) a mixture of dimethylforrnamide and ahalogenating agent to produce 2, 2-cyclocytidine or said salt thereofand then hydrolyzing the 2, 2'- cyclocytidine or salt by mixing it withwater and adjusting the pH of the mixture to a value from about 9 to 12.

2. A process according to claim '1 for preparing 1B-D-arabinofuranosylcytosine or a salt thereof wherein the halogenatingagent is at least one substance selected from the group consisting ofthionyl chloride, thionyl bromide, phosphorus oxychloride, phosphorusoxybromide, phosphorus trichloride, phosphorus tribromide, phosgene andchlorine.

3. A process according to claim 1 for preparing 1-[3-arabinofuranosylcytosine or said salt thereof wherein the halogenatingagent is phosphorus pentachloride, phosphorus pentabromide or acombination thereof.

4. A process for preparing l-B-D- arabinofuranosylcytosine or aninorganic acid salt or organic acid salt thereof consisting essentiallyof reacting cytidine, a mineral acid salt thereof, or a cytidine salt ofan acid selected from the group consisting of formic acid, acetic acid,sulfosalicyclic acid and erotic acid, with at least one substanceselected from the group consisting of diphalophosphoric acid, phosphorusoxyhalides and partially hydrated phosphorus oxyhalides to produce 2, 2cyclocytidine or said salt thereof and then hydrolyzing the 2,ZI-cyclocytidine or salt by mixing it with water and adjusting the pH toa value from about 9 to 12.

5. A process according to claim 4 for preparing l-B-D-arabinofuranosylcytosine or said salt thereof wherein an acetic acidester is used as the reaction solvent.

6. A process for preparing l 48-D- arabinofuranosylcytosine or aninorganic acid salt or organic acid salt thereof consisting essentiallyof react- I ing cytidine, a mineral acid salt thereof, or a cytidinesalt of an acid selected from the group consisting of formic acid,acetic acid, sulfosalicyclic acid and crotic acid, in an acetic acidester as solvent with at least one substance selected from the groupconsisting of (a) dichlorophosphoric acid, (b) phosphorous oxychloride,(c) phosphorus oxybromide, (d) partially hydrated phosphorus oxychlorideand (e) partially hydrated phosphorus oxybromide to produce 2,2'-cyclocytidine or said salt thereof and then hydrolyzing the 2, 2'-cyclocytidine or said salt by mixing it with water and adjusting the pHof the mixture to a value from about 9 to 12.

7. A process according to claim 6 for preparingl-B-D-arabinofuranosylcytosine or said salt thereof wherein phosphorusoxychloride or phosphorus oxybromide is added as a mixture with atertiary alcohol to react with cytidine or said salt thereof.

8. A process according to claim 7 for preparing l-B-D-arabinofuranosylcytosine or said salt thereof wherein the mentionedtertiary alcohol is tert-butanol.

9. A process according to claim 1 for preparing l-B-D-arabinofuranosylcytosine or said salt thereof comprising directlyhydrolyzing the 2,2'-cyclocytidine or said salt thereof as anintermediate without isolating the same from the reaction solution.

10. A process for preparing 2, 2'-cyclocytidine or an inorganic acidsalt or organic acid salt thereof consisting essentially of reactingcytidine, a mineral salt thereof, or a cytidine salt of an acid selectedfrom the group consisting of formic acid, acetic acid, sulfosalicyclicacid and erotic acid, with at least one material selected from the groupconsisting of (a) a halomethylenedimethylammonium halide expressed bythe general formula where X, and X, stand for halogen atoms of the sameor different species, and (b) a mixture of dimethylformamide and ahalogenating agent.

11. A process according to claim 10 for preparing 2,2'-cyclocytidine orsaid salt thereof wherein the halogenating agent is at least onesubstance selected from the group consisting of thionyl chloride,thionyl bromide, phosphorus oxychloride, phosphorus oxybromide,phosphorus trichloride, phosphorus tribromide, phosgene and chlorine.

12. A process according to claim 10 for preparing 2,2'-cyclocytidine orsaid salt thereof wherein the halogenating agent being is phosphoruspentachloride or phosphorus pentabromide.

13. A process for preparing 2, 2'-cyclocytidine or an inorganic acidsalt or organic acid salt thereof consisting essentially of reactingcytidine, a mineral acid salt thereof, or a cytidine salt of an acidselected from the group consisting of formic acid acetic acid,sulfosalicylic acid and erotic acid, with at least one substanceselected from the group consisting of dihalophosphoric acids, phosphorusoxyhalides and partially hydrated phosphorus oiiyhalides.

14. A process according to claim 13 for preparing 2,2-cyclocytidine orsaid salt thereof wherein an ester of acetic acid is used as thereaction solvent.

15. A process for preparing 2, 2-cyclocytidine or an inorganic acid saltor organic acid salt thereof consisting essentially of reactingcytidine, a mineral acid salt thereof, or a cytidine salt of an acidselected from the group consisting of formic acid, acetic acid,sulfosalicylic acid and erotic acid, in an ester of acetic acid assolvent with at least one substance selected from the group consistingof (a) dichlorophosphoric acid, (b) phosphorus oxychloride, (c)phosphorus oxybromide, (d) partially hydrated phosphorus oxychloride and(c) partially hydrated phosphorus oxybromide.

16. A process according to claim 15 for preparing 2,2'-cyclocytidine ora said salt thereof comprising applying phosphorus oxychloride orphosphorus oxybromide in the form of a mixture with a tertiary alcoholto react with cytidine or a said salt thereof.

17. A process according to claim 16 for preparing 2,2'-cyclocytidine orsaid salt thereof wherein the tertiaryalcohol is ter t-butanol.

2. A process according to claim 1 for preparing 1 Beta-D-arabinofuranosylcytosine or a salt thereof wherein the halogenatingagent is at least one substance selected from the group consisting ofthionyl chloride, thionyl bromide, phosphorus oxychloride, phosphorusoxybromide, phosphorus trichloride, phosphorus tribromide, phosgene andchlorine.
 3. A process according to claim 1 for preparing 1- Beta-arabinofuranosylcytosine or said salt thereof wherein the halogenatingagent is phosphorus pentachloride, phosphorus pentabromide or acombination thereof.
 4. A process for preparing 1- Beta-D-arabinofuranosylcytosine or an inorganic acid salt or organic acidsalt thereof consisting essentially of reacting cytidine, a mineral acidsalt thereof, or a cytidine salt of an acid selected from the groupconsisting of formic acid, acetic acid, sulfosalicyclic acid and croticacid, with at least one substance selected from the group consisting ofdiphalophosphoric acid, phosphorus oxyhalides and partially hydratedphosphorus oxyhalides to produce 2, 2'' cyclocytidine or said saltthereof and then hydrolyzing the 2, 2''-cyclocytidine or salt by mixingit with water and adjusting the pH to a value from about 9 to
 12. 5. Aprocess according to claim 4 for preparing 1- Beta-D-arabinofuranosylcytosine or said salt thereof wherein an acetic acidester is used as the reaction solvent.
 6. A procesS for preparing 1-Beta -D-arabinofuranosylcytosine or an inorganic acid salt or organicacid salt thereof consisting essentially of reacting cytidine, a mineralacid salt thereof, or a cytidine salt of an acid selected from the groupconsisting of formic acid, acetic acid, sulfosalicyclic acid and croticacid, in an acetic acid ester as solvent with at least one substanceselected from the group consisting of (a) dichlorophosphoric acid, (b)phosphorous oxychloride, (c) phosphorus oxybromide, (d) partiallyhydrated phosphorus oxychloride and (e) partially hydrated phosphorusoxybromide to produce 2, 2''-cyclocytidine or said salt thereof and thenhydrolyzing the 2, 2''-cyclocytidine or said salt by mixing it withwater and adjusting the pH of the mixture to a value from about 9 to 12.7. A process according to claim 6 for preparing 1- Beta-D-arabinofuranosylcytosine or said salt thereof wherein phosphorusoxychloride or phosphorus oxybromide is added as a mixture with atertiary alcohol to react with cytidine or said salt thereof.
 8. Aprocess according to claim 7 for preparing 1- Beta-D-arabinofuranosylcytosine or said salt thereof wherein the mentionedtertiary alcohol is tert-butanol.
 9. A process according to claim 1 forpreparing 1- Beta -D-arabinofuranosylcytosine or said salt thereofcomprising directly hydrolyzing the 2,2''-cyclocytidine or said saltthereof as an intermediate without isolating the same from the reactionsolution.
 10. A process for preparing 2, 2''-cyclocytidine or aninorganic acid salt or organic acid salt thereof consisting essentiallyof reacting cytidine, a mineral salt thereof, or a cytidine salt of anacid selected from the group consisting of formic acid, acetic acid,sulfosalicyclic acid and crotic acid, with at least one materialselected from the group consisting of (a) ahalomethylenedimethylammonium halide expressed by the general formula11. A process according to claim 10 for preparing 2,2''-cyclocytidine orsaid salt thereof wherein the halogenating agent is at least onesubstance selected from the group consisting of thionyl chloride,thionyl bromide, phosphorus oxychloride, phosphorus oxybromide,phosphorus trichloride, phosphorus tribromide, phosgene and chlorine.12. A process according to claim 10 for preparing 2,2''-cyclocytidine orsaid salt thereof wherein the halogenating agent being is phosphoruspentachloride or phosphorus pentabromide.
 13. A process for preparing 2,2''-cyclocytidine or an inorganic acid salt or organic acid salt thereofconsisting essentially of reacting cytidine, a mineral acid saltthereof, or a cytidine salt of an acid selected from the groupconsisting of formic acid acetic acid, sulfosalicylic acid and croticacid, with at least one substance selected from the group consisting ofdihalophosphoric acids, phosphorus oxyhalides and partially hydratedphosphorus oxyhalides.
 14. A process according to claim 13 for preparing2,2''-cyclocytidine or said salt thereof wherein an ester of acetic acidis used as the reaction solvent.
 15. A process for preparing 2,2''-cyclocytidine or an inorganic acid salt or organic acid salt thereofconsisting essentially of reacting cytidine, a mineral acid saltthereof, or a cytidine salt of an acid selected from the groupconsisting of formic acid, acetic acid, sulfosalicylic acid and croticacid, in an ester of acetic acid as solvent with at least one substanceselected from the group consisting of (a) dichlorophosphoric acid, (b)phosphorus oxychloride, (c) phosphorus oxybromide, (d) partiallyhydrated phosphorus oxychloride and (e) partially hydrated phosphorusoxybromide.
 16. A process according to claim 15 for preparing2,2''-cyclocytidine or a said salt theReof comprising applyingphosphorus oxychloride or phosphorus oxybromide in the form of a mixturewith a tertiary alcohol to react with cytidine or a said salt thereof.17. A process according to claim 16 for preparing 2,2''-cyclocytidine orsaid salt thereof wherein the tertiary alcohol is tert-butanol.